Randomised mutagenesis of genes of interest has recently deserved increased attention. In the existing prior art, a library of mutated genes is first generated and then screened for the presence of mutants possessing a given property either in vitro or, if possible, through genetic screening in bacterial cells. However, when eukaryotic proteins are targeted, the properties observed in vitro for a given mutant often do not result in the desired phenotype when introduced in the eukaryotic cell. For example, it has been shown that mutants isolated for their ability to carry out a specific enzymatic activity, as the phosphorylation of thymidine residues or thymidine kinase (TK) activity in vitro or in bacteria, proved to be inefficient to confer a TK+ phenotype to TK-human cells in culture.
To date, only one system, the tetracycline-regulated gene expression system (Tet system), that benefits from the propensity of retroviruses to promote genetic diversity for molecular evolution experiments has been demonstrated (Das A. T., Zhou X., Vink M., Klaver B., Verhoef K., et al. (2004) J. Biol. Chem., 279: 18776-82). In Das et al., replication-competent viruses (functional viruses) were constructed, and genomic expression was driven by the Tet system. By selecting for the faster replicating variants, an improved Tet system was selected. However, in the approach adopted by Das et al., selection was necessarily coupled to viral replication capacity, and its applications extremely limited.
However, it is also known in the art that the use of functional viruses, and more particularly human immunodeficiency virus (HIV), presents several drawbacks, the most important of which are (a) the cytotoxicity of viral infection that constitutes a major obstacle to subsequent selection of cells with the desired phenotype, (b) the limited availability of “free” room in the viral genome to harbour exogenous genes, and (c) the impossibility of blocking the infectious process when desired.
Another approach is to generate a bank of mutated genes by degenerated polymerase chain reaction (PCR), followed by insertion of the thereby generated library in bacteria. A common step after the generation of the library is then the screening for the mutant possessing the desired phenotype, generally in vitro or, when possible, in bacteria having the appropriate genetic background for such screening. However, when the mutated gene is intended to confer a desired phenotype to a human cell, these pre-screening tests often lead to the isolation of mutants that actually do not confer the desired phenotype to the human cell. Direct screening of the library in the human cell by inserting the genes by transfection is also made difficult by the low efficiency of generation of stable clones by this method, which dramatically reduces the complexity of the library, and by the problem that this procedure generates, of the insertion of multiple copies of the gene in the cell genome. This hampers clonal screening. Insertion of the library in lentiviral vectors allows clonal screening quite easily, instead. However, transposing the library from the bacteria to the lentiviral vectors also suffers from a drastic low efficiency that reduces the complexity of the library. Generating the library directly in these vectors, instead, bypasses this problem.
Therefore, there is a need for a system or a method for molecular evolution that avoids at least some of these drawbacks. This is the purposes of the method hereafter disclosed.